Optimization of high cell density fermentation process for recombinant nitrilase production in E. coli

Area of Science:

• Biocatalysis and enzyme engineering
• Microbial fermentation technology
• Protein expression and optimization

Background:

• Nitrilases are key biocatalysts for producing chiral compounds.
• Efficient production of nitrilases is essential for industrial applications.
• Escherichia coli is a well-established host for recombinant protein expression.

Purpose of the Study:

• To optimize nitrilase production in Escherichia coli BL21 (DE3).
• To enhance high cell density fermentation for improved enzyme yield.
• To investigate process parameters affecting Pseudomonas fluorescens nitrilase expression.

Main Methods:

• High cell density fermentation in batch and fed-batch modes.
• Optimization of temperature, substrates, and inducer (IPTG) concentrations.
• Utilizing a T7 promoter-based expression system in E. coli.

Main Results:

• Super optimal broth with glycerol without inducer yielded best results in batch mode.
• Optimal temperature for batch production was determined to be 32 °C.
• Isopropyl β-D-1-thiogalactopyranoside (IPTG) induction resulted in insoluble protein and reduced enzyme activity.
• Optimized fed-batch strategy led to a 40-fold increase in volumetric enzyme activity.

Conclusions:

• Fed-batch fermentation is superior to batch for high-yield nitrilase production.
• Avoiding IPTG induction improves enzyme solubility and activity in this system.
• The optimized process significantly enhances the potential of nitrilases in chiral synthesis.